Staphylococcus aureus is one of the most common causes of acute and chronic infections in both community and hospital settings. As an innocuous commensal, S. aureus colonizes a large percentage of the healthy adult population, predominantly in the nasal passages, but often transitions into a virulent pathogen, disseminating and causing severe and devastating disease. The S. aureus cell-to-cell communication system, also called quorum-sensing or the Agr system, is thought to be important in the switch to an invasive state, but how this transition occurs when S. aureus is in a biofilm or inside a neutrophil is not clear. We propose that quorum-sensing controls a universal dispersal mechanism allowing S. aureus to transition out of a biofilm state and evade host defenses. In support of this hypothesis, we developed a novel method to synthesize the quorum-sensing signal and found that Agr activation enables detachment from biofilms. Further, our preliminary results have uncovered that up- regulation of the Agr regulon precedes detachment and that the mechanism is mediated by extracellular serine proteases. In this application, we propose to define the biofilm detachment mechanism. For Specific Aim 1, we will (i) perform microarray experiments to identify genes up and down-regulated prior to detachment; (ii) identify the serine proteases mediating biofilm detachment; and (iii) characterize the identified detachment enzymes. In preliminary tests, we have also gained expertise in growing S. aureus at the air-liquid interface of human airway epithelial cells, and we hypothesize that quorum-sensing activation will modulate attachment and detachment from these epithelial cells. To address this question, in Specific Aim 2, we will (i) define the role of quorum- sensing in attachment and biofilm formation on airway epithelia; (ii) determine the epithelia inflammatory responses; and (iii) investigate detachment from epithelial cells. Finally, activation of quorum-sensing is known to precede S. aureus escape from non-phagocytic cells. Whether a similar mechanism occurs with professional phagocytes, such as polymorphonuclear leukocytes (PMN), is not clear. We hypothesize that quorum-sensing activation will precede escape from PMN, and we have performed proof-of-concept studies to begin addressing this question. Towards this end, in Specific Aim 3, we will (i) examine interactions between PMN and quorum-sensing active and inactive strains; (ii) investigate the impact of S. aureus quorum-sensing in susceptibility to synergistic killing by PMN and group IIA phospholipase A2; and (iii) analyze PMN interactions with S. aureus cells that have detached from airway epithelia or escaped phagosomes. In each specific aim, we will also compare the transition mechanisms of laboratory strains with the community-associated methicillin resistant strains (CA-MRSA), in order to gain insight into the contribution of quorum-sensing to the exceptional virulence of CA-MRSA. Altogether, the results of these studies will expand our understanding of the S. aureus mechanisms used to transition from a colonizer into an invader. Knowledge of these transition mechanisms could lead to innovative therapies that block the spread of this virulent, invasive pathogen. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus normally lives in the human nasal passages without incident, and yet when presented with an opportunity, this pathogen is one of the most common causes of bacterial infections in both community and hospital settings. Our studies will advance the understanding of how S. aureus uses a cell-to-cell communication system to control the transition from a variety of lifestyle states into an invasive pathogen.